BRIEF NOTES 335

involved in the regulation of food intake and lipid metabolism asseveral different murine agouti alleles are associated with obesity inaddition to the colour variations1. We have isolated three genomicPAC clones2 with the ASIP gene from the pig and determined itscomplete genomic organization. As in other mammalian species, itconsists of four exons, which encode a cDNA of about 700 bp3–6.The first untranslated exon is separated by a large intron from theother three coding exons, which are contained within 5 kb ofgenomic DNA (Fig. 1, Table 1). The encoded 131 amino acid proteinshows 82% identity to the human ASIP. The nucleotide sequence ofthe porcine ASIP gene has been deposited in the EMBL nucleotidedatabase under accession numbers AJ251836 and AJ251837. Com-parison of ASIP genomic sequences from different pig breedsrevealed the presence of two polymorphic sites in intron 1 and onepolymorphic site in the 3% untranslated region of exon 4.

Primer sequences:pASIP–1fw: 5%-ACCTAAACGATCCAGAAAC-3%pASIP–1rev: 5%-CCACCCTTAATTACCCAG-3%pASIP–2fw: 5%-TCTTCCTTCATCAGTTCCC-3%pASIP–2rev: 5%-TTTAACATCCTGCAGCCC-3%pASIP–4fw: 5%-GAGAAGAGGGCCAAATCG-3%pASIP–4rev: 5%-GGAAAGGGTGAAAGGCTG-3%

PCR conditions: PCR reactions were performed in a total volume of100 ml containing 100 ng template DNA, 100 pmol of each primer,200 mM dNTPs, and 2 U Taq polymerase (Qiagen, Hilden, Germany)in the buffer supplied by the manufacturer. Following a 5 mininitial denaturation at 94 °C, 32 cycles of 1 min at 94 °C, 1 min at54 °C, and 1 min at 72 °C were performed in a Hybaid OmnigeneThermal Cycler (MWG-Biotech, Eberberg, Germany). For sequenc-ing one PCR primer of each primer pair was tailed with the 18 ntUniversal sequence. The resulting PCR products were sequencedwith the thermosequenase kit (AmershamPharmacia, Freiburg, Ger-many) using IRD-labelled universal primer on a LICOR 4000Lautomated sequencer.

PCR conditions for mapping : PCR reactions performed separatelyfor dog and hamster DNAs were used as controls. The reactionconsisted of 1 cycle of 95 °C for 2 min, followed by 30 cycles of95 °C for 20 s, 60 °C for 40 s, 72 °C for 1 min, and final extension at72 °C for 3 min.

Mendelian segregation : We confirmed codominant segregation ofthe polymorphic marker in a number of pedigree of Korean Sapsa-ree (Fig. 2).

Acknowledgements: This study was supported by Creative Re-search Initiative Program to C. Park.

References1 Kim K.S. et al. (1998) Mol Phylogenet Evol 10, 210–20.2 Das M. et al. (1998) Mamm Genome 9, 64–9.3 Bentolila S. et al. (1999) Mamm Genome 10, 699–705.4 Mellersh C.S. et al. (2000) Mamm Genome 11, 120–30.

Correspondence: C Park

Genomic structure and nucleotidepolymorphisms of the porcine agouti signallingprotein gene (ASIP)T Leeb1,2, A Deppe1, B Kriegesmann1, B Brenig1

1 Institute of Veterinary Medicine, University of Gottingen,Germany; 2 Present address: Institute of Animal Breeding, Schoolof Veterinary Medicine Hannover, Buenteweg 17p, 30559Hannover, Germany

Accepted 9 May 2000

Source/description: The agouti signalling protein (ASIP) has animportant function in the regulation of coat colour. It might also be

Fig. 1. Genomic structure of the porcine ASIP gene. Thin lines indicate non-coding regions. Exons are shown as boxes and are numbered. Coding regionsare shown as closed boxes whereas 5% and 3% untranslated regions are shown as cross-hatched boxes. The three polymorphic sites are indicated. Arrowsdenote the position of the primers for amplification of these polymorphisms. The DNA sequences of the illustrated region have been deposited in theEMBL database, Acc. AJ251836 and AJ251837.

Table 1. Exon-intron junctions of the porcine ASIP gene

Exon (bp) Splice-acceptorIntron (bp)Splice-donorNo.

−10−11:20 kb ...tctcactcagGCCTCCA1 \90 CAGAGAGgtatgtaata...

160 161GTG Ggtaagcagcc... ...atctttgaagCA CTG12691702Val A la Leu

219 2203161TCC AAGgtaggcctgg... ...ttccccacagAAA AAG593

Lys LysSer Lys589

\370 AAATTCCAAATACACGC...4

Exon sequences are shown in uppercase letters, intron sequences in lowercase letters and untranslated regions in italics. The conserved GT/AGexon/intron-junctions are shown in bold. For the last exon the putative polyadenylation signal is shown in bold italics instead of a splice donor. Amino acidresidues are indicated with respect to each boundary. Numbers refer to the corresponding positions in the porcine ASIP cDNA starting with +1 at theadenine of the start codon ATG.

© 2000 International Society for Animal Genetics, Animal Genetics 31, 333–346

BRIEF NOTES336

Table 2. Polymorphisms in the porcine ASIP sequence

cDNA NucleotideLocation Nucleotidepositionposition polymorphism

C6�C7394–399a)intron 1intron 1 1107b) A�G

T�C+508c)6128b)exon 4(3%-UTR)

a) numbering refers to EMBL accession AJ251836.b) numbering refers to EMBL accession AJ251837.c) +1 corresponds to the adenine of the startcodon ATG of the ASIP cDNA.

Fig. 1. Photograph of an ethidium bromide stained gel (4%) of an embryotransfer family from a Limousin sire homozygous for the cut allele and aheterozygous Belgian Blue dam. The uncut allele is 79 bp and the cutallele is 61 bp. A constant band is also evident at 58 bp.

Polymorphisms: Sequence analysis of the four exons with flankingregions in nine pigs from different breeds (German Landrace,German Large White, Pietrain, Angler Saddleback, Duroc, BunteBentheimer, and wild boar) revealed three polymorphisms, whichare all located in non-coding parts of the porcine ASIP gene (Table2). One of these polymorphisms affects the length of a polyC stretchin intron 1 while the other two sequence variations represent singlenucleotide polymorphisms (SNPs).

Chromosomal location : The porcine ASIP gene was mapped re-cently by FISH analysis and analysis of a somatic cell hybrid panelto SSC17q217.

References1 Lu D. et al. (1994) Nature 371, 799–802.2 Al-Bayati H.K. et al. (1999) Mamm Genome 10, 569–72.3 Bultman S.J. et al. (1992) Cell 71, 1195–204.4 Vage D.I. et al. (1997) Nat Genet 15, 311–5.5 Kwon H.Y. et al. (1994) Proc Natl Acad Sci USA 91, 9760–4.6 Wang Y. et al. (1998) Pigment Cell Res 11, 155–7.7 Kijas J.M.H. et al. (1998) Chromosome Res 6, 243.

Correspondence: Dr Tosso Leeb (e-mail:tleeb@zucht.tiho-hannover.de)

An SNP in DCT is used for linkage mapping oncattle chromosome 12S M Schmutz, T G Berryere, F C BuchananDepartment of Animal and Poultry Science, University ofSaskatchewan, Saskatoon, Saskatchewan, Canada S7N 5B5

Accepted 22 May 2000

Source/description: Dopachrome tautomerase (DCT) (dopachromedelta-isomerase, tyrosine-related protein 2) is the gene referred toas slaty in the mouse1 which is thought to dilute eumelanin but notphaeomelanin2. We were interested in finding a polymorphismwithin this gene because it is part of the colour pigment pathwaywithin the melanocyte. A yeast artificial chromosome (YAC) clonecontaining DCT was previously mapped by in situ hybridization tochromosome 12q233. Primer sequences3 were used to amplify aDNA fragment (Genbank U46153) equivalent to exon 2, based onthe human sequence, from three cattle. Two single nucleotidepolymorphisms (SNPs) were identified at bp 28 and 91 of theproduct (Genbank AF152005; nt 24 and 87, respectively), but norestriction enzyme was found to detect either mutation. Therefore,we designed a purposeful mismatch forward primer that intro-duced an AvaII cut site at the first mutation site and used thepreviously published reverse primer3.

Primer sequences:Forward: TAG ACC TCG CAA AGT ACG GTC

Reverse: ATA ATG GAG CCA CAC AAA GAA A

PCR conditions : The PCR reaction mixture of 15 ml contained 1 mlDNA (50–100 ng), 1·5 ml PCR buffer (Gibco, BRL, Burlington, ON,Canada), 0·45 ml 50 mM MgCl2, 1 ml 10 mM dNTP, 0·1 ml Taqpolymerase (Gibco) and 1 ml (10 pM/ml) of each primer. The cycling

protocol was 4 min at 95 °C, continued for 30 cycles of 30 s at 95 °C,30 s at 57 °C, 15 s at 72 °C and finished with a 4 min extension at72 °C. Digestion with AvaII was carried out for 3 h at 37 °C.Polymorphism: The 137-bp product was either cut into 79 and 58bp fragments or into 61, 58 and 18 bp fragments. Both alleles weredetected in the homozygous and heterozygous states after AvaIIdigestion. The 61-bp allele occurred with a frequency of 32% in the23 unrelated cattle of the parental generation of the Canadian BeefCattle Reference Herd.Mendelian inheritance : Segregation consistent with codominantinheritance was found in six families, as shown in Fig. 1.Chromosomal location : The Canadian Beef Cattle Reference Herd:17 cattle families are full-sib families from five sires and 16 dams(Angus, Belgian Blue, Charolais, Hereford, Limousin, Simmental)(http://sask.usask.ca/�schmutz) were used for linkage mapping.As part of the larger quantitative trait loci (QTL) study, 162microsatellites, six of which were on cattle chromosome 12, wereused to genotype all 18 parents and 136 offspring. DCT wasmapped 5 cM from INRA005 (LOD=3·092), which is 83 cM fromthe centromere on the USDA (United States Department fo Agricul-ture) map of cattle chromosome 124. It appears to be about 10 cM

from BMS2724 (LOD=3·85), which is the most telomeric marker at105 cM on the USDA map4. The most likely order using CRI-MAP was BMS410-BM6116-ILSTS010-BMS975-DCT-INRA005-BMS2724. DCT maps to human chromosome 13q31-q325, to mousechromosome 141, and to pig chromosome 116. These comparativemapping data are consistent with DCT mapping to cattle chromo-some 12.Comment: EDNRB, another coat colour gene, was also mapped tocattle chromosome 12q22 by in situ hybridization7. Smoke greycattle are often the result of black Angus crossed with whiteCharolais cattle. Although no association was found between thisSNP and coat colour, no slate grey animals occurred in our herd;thus, this does not rule out a possible association with that coatcolour in cattle.Acknowledgements: The Canadian Beef Reference Herd was raisedthrough joint funding from the Canadian Cattlemen’s Association,the Alberta Cattle Commission and the Natural Science and Engi-neering Research Council–Industry Oriented Research program.We thank the staff at the University of Saskatchewan beef facilitiesand all of the students who helped with this herd.References1 Jackson I.J. et al. (1992) EMBO 11, 527–35.2 Budd P., Jackson I.J. (1995) Genomics 29, 35–43.3 Hawkins G.A. et al. (1996) Mamm Genome 7, 474–5.4 Kappes S.M. et al. (1997) Genome Res 7, 235–49.5 Sturm R.A. et al. (1994) Genomics 21, 293–6.6 Chowdhary B.P. et al. (1993) Chromosome Res 1, 175–9.7 Schlapfer J. et al. (1997) Mamm Genome 8, 380–1.

Correspondence: S M Schmutz (e-mail: schmutz@sask.usask.ca)

© 2000 International Society for Animal Genetics, Animal Genetics 31, 333–346